UGANDA STANDARD  
      US ISO 6493
First Edition
   2009 -09-04
 
Reference numbe r 
   US ISO 6493:2000
 © UNBS 200 9 
  
Animal feeding stuffs — Determination of starch content—
Polarimetric method 
 US ISO 6493:2000  
 
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    US ISO 6493:2000  
© UNBS  2009 - All rights reserved National foreword 
Uganda National Bureau of Standards (UNBS) is a parastatal under the Ministry  of Tourism, Trade and 
Industry established by the Act of Parliament of 198 3, of the Laws of Uganda.  UNBS is mandated to co-
ordinate the elaboration of standards and is  
(a) a member of International Organisation for Standardisation (ISO) and  
(b) a contact point for the WHO/FAO Codex A limentarius Commission on Food Standards, and  
(c) the National Enquiry Point on TBT/SPS Agr eements of the World Trade Organisation (WTO). 
The work of preparing Uganda Standards is carried out through Technical Committees. A Technical 
Committee is established to deliberate on standards in a gi ven field or area and consists of representatives of 
consumers, traders, academician s, manufacturers, governme nt and other stakeholders. 
Draft Uganda Standards adopted by the Technical Committ ee are widely circulated to stakeholders and the 
general public for comments. The committee reviews the comments before recommending the draft standards 
for approval and declaration as Uganda Standards by the National Standards Council. 
This Uganda Standard, US ISO 6493:2000, Animal feeding stuffs — Determ ination of starch content —
Polarimetric method, is identical with and  has been reproduced from an International Standard ISO
6493:2000,  Animal feeding stuffs —Determination of starch content — Polarimetric method, and has been
adopted as a Uganda Standard . 
Wherever the words, "International Standard" a ppear, they should be replaced by "Uganda Standard." Reference number
ISO6493:2000(E)
©ISO2000INTERNATIONAL
STANDARDISO
6493
Firstedition
2000-02-01
Animal feedingstuffs— Determinationof
starchcontent— Polarimetric method
Aliments des animaux — Déterminationdelateneur enamidon —
Méthode polarimétriqueISO 6493:2000(E)
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ii © ISO2000 –All rights reservedISO 6493:2000(E)
© ISO2000 –All rights reserved iiiForeword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO
member bodies). The work of preparing International Standards is normally carried out through ISO technical
committees. Each member body interested in a subject for which a technical committee has been established hasthe right to be represented on that committee. International organizations, governmental and non-governmental, inliaison with ISO, also take part in the work. ISO collaborates closely with the International ElectrotechnicalCommission(IEC) onall matters of electrotechnicalstandardization.
InternationalStandards are drafted in accordance with the rules givenintheISO/IEC Directives, Part3.
Draft International Standards adopted by the technical committees are circulated to the member bodies for voting.
Publicationas anInternationalStandardrequires approval byat least 75% of themember bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this International Standard may be the subject of
patent rights. ISO shall not beheldresponsiblefor identifyinganyor allsuch patent rights.
International Standard ISO6493 was prepared by Technical Committee ISO/TC34,
Agricultural food products ,
SubcommitteeSC 10, Animalfeedingstuffs .
Annexes A and B of this International Standardare for information only.INTERNATIONAL STANDARD ISO 6493:2000(E)
© ISO2000 –All rights reserved 1Animal feeding stuffs —Determination ofstarch content —
Polarimetricmethod
1 Scope
This International Standard specifies a method for the polarimetric determination of the starch content of animal
feeding stuffs andraw materials for animalfeedingstuffs.
Themethod is not applicable to products which, besides starch, contain other substances which are opticallyactive
during the analysis and do not dissolve in40% ethanol. Examples of these products are potato pulp, beet chips,beet leaves, beet tops, yeast, soya products, lupine and products rich in inulin (e.g. chicory roots and Jerusalem
artichokes). Inthese cases an enzymatic determinationof thestarchcontent maybe used.
This method is not applicable for the quantification of starch with an amylose content exceeding 40% (e.g. high
amylose maize starch like HylonVII).
CAUTION — Dependingontheintensityof theheat/moisturetreatment theproduct has been subjected to,
toolow astarchcontent maybedetermined.
2 Normative references
The following normative documents contain provisions which, through reference in this text, constitute provisions of
this International Standard. For dated references, subsequent amendments to, or revisions of, any of these
publications do not apply. However, parties to agreements based on this International Standard are encouraged toinvestigate the possibility of applying the most recent editions of the normative documents indicated below. Forundated references, the latest edition of the normative document referred to applies. Members of ISO and IECmaintain registers of currentlyvalid InternationalStandards.
ISO 3310-1,
Test sieves — Technicalrequirements andtesting— Part1: Test sieves of metalwirecloth .
ISO 3696, Water for analyticallaboratory use— Specification and test methods .
ISO 6498, Animalfeedingstuffs — Preparation of test samples .
3 Terms anddefinitions
For the purposes of this International Standard, thefollowing terms anddefinitions apply.
3.1
starch
natural vegetable polymer consisting of long unbranched chains of /G61-1,4-linked glucose-units (amylose) and/or
long/G61-1,6-branchedchains of /G61-1,4-linkedglucose-units(amylopectine)ISO 6493:2000(E)
2 © ISO2000 –All rights reserved3.2
starch content
mass fraction of starch and its high molecular mass breakdown products, insoluble in40% ethanol, and
determined inaccordancewith this International Standard
NOTE The starch content is expressed in gramsperkilogram.
4P r i n c i p l e
A test portion is decomposed with dilute hydrochloric acid, then the solubilized starch is gelatinized and partiallyhydrolysed.
Thetotal opticalrotation of theclarified solution is determined.Correction is made for the optical rotation caused by other substances which are soluble in 40% ethanol and
opticallyactiveafter treatment with dilutehydrochloric acid.
Thestarchcontent is calculated bymultiplyingthecorrectedopticalrotationbya knownfactor.
5 Reagents
Useonlyreagents of recognized analyticalgrade.
5.1 Water , complying with at least grade3 inaccordancewithISO3696.
5.2 Ethanol (C2H5OH), 40% volumefraction.
5.3 Methylred , solution in ethanol(96% volumefraction), /G72(methylred)=1g/l.
5.4 Hydrochloricacid ,c(HCl)=0,31 mol/l.
Check the concentration by titration with a 0,100mol/l sodium hydroxide solution using methyl red as indicator. An
aliquot portionof 10ml of hydrochloric acidshallneutralize(31,0 /Gb10,1)mlof thesodium hydroxidesolution.
CAUTION — Too high or too low a hydrochloric acid concentration will lead to incorrect values of the
determined starch content.
5.5 Hydrochloricacid ,c(HCl)=7,73 mol/l.
5.6 Clarifying solutions ,according toCarrez, asfollows.
5.6.1 Potassium hexacyanoiron(II) solution ,c[K4Fe(CN)6] =0,25 mol/l.
In a 1l volumetric flask, dissolve 106g of potassium hexacyanoiron(II) trihydrate [K4Fe(CN)6/Gd73H2O] in water. Dilute
tothemark with water.
5.6.2 Zincacetate, solution in0,5 mol/l acetic acid, c[Zn(CH3CO2)2]=1mol/l.
In a 1l volumetric flask dissolve 219,5g of zinc acetate dihydrate [Zn(CH3CO2)2/Gd72H2O] and 30g of glacial acetic
acid in water. Dilute tothemark with water.
5.7 Saccharosesolution ,/G72(C12H22O11)=100,0g/l.ISO 6493:2000(E)
© ISO2000 –All rights reserved 36 Apparatus
Usual laboratoryapparatus and, in particular, thefollowing.
6.1 Analyticalbalance , capable of weighingtothenearest 1mg.
6.2 pH-meter , capableof measuringtothenearest 0,1pHunit.
6.3 Boiling water bath , with sufficient capacity to maintain the water at boiling point while immersing the conical
flasks.
CAUTION — If thewaterbath isnot boiling constantly,too high astarch content willbedetermined.
6.4 Polarimeter ,accurate to atleast0,01°andsuitablefor 200mm longtubes.
Measure the optical rotation at a wavelength of 589,3nm (sodium D line). When using polarimeter tubes of
deviatinglengths, measurewith acorresponding accuracy.
A saccharimeter may be used if it has an accuracy of measurement at least equal to the accuracy of the
polarimeter. Inthis case, convert thereadings todegrees.
The polarimeter may be calibrated with the saccharose solution (5.7). This saccharose solution yields an optical
rotationof 13,30° whenmeasuredat (20 /Gb11)/Gb0C usinga200mm polarimeter tube.
6.5 Burette .
6.6 Refluxcondensers .
6.7 Volumetricflasks , of capacity100ml.
If the volumetric flasks need to be fitted to a reflux condenser (see9.3.3), the use of wide-necked Kohlrausch
conical flasks is recommended.
NOTE Kohlrausch conicalflasks are commerciallyavailable volumetric flasksused forsugardeterminations.
7 Sampling
Sampling is not part of the method specified in this International Standard. A recommended sampling method is
given in ISO 6497 [1].
It is important that the laboratory receive a sample which is truly representative and has not been damaged or
changedduringtransport or storage.
8 Preparationoftestsample
Prepare the test sampleinaccordance withISO6498.
If solid, grind the laboratory sample (usually 500g) so that it passes completely through a sieve with 0,5mm
apertures complying withISO3310-1. Mix thoroughly.ISO 6493:2000(E)
4 © ISO2000 –All rights reserved9 Procedure
9.1Determination of acid consumption
9.1.1Weigh approximately2,5g of the prepared test sample to the nearest 1mg and transfer it quantitatively to a
50mlconicalflask. Add 25ml of water andshakeuntila homogeneous suspension is obtained.
9.1.2Place the electrodes of the pH-meter (6.2) in the suspension and, using a burette, add hydrochloric acid
(5.4) until the pH of the suspension is 3,0 /Gb10,1. Check whether the complete acid consumption of the test portion
has been compensated for, by shaking the suspension vigorously and allowing it to stand for 2min. If during thisperiod the pH has increased up to a value exceeding 3,1, add more hydrochloric acid (5.4), using the burette, as
manytimes as necessaryuntil nomoreacid is consumed.
9.1.3Calculatetheacidconsumptionof the test portionfrom thevolumeof hydrochloric acid (5.4) added.
9.2Determinationoftotal opticalrotation
9.2.1Weigh approximately2,5g of the prepared test sample ( m1) to the nearest 1mg and transfer it quantitatively
to a dry100ml volumetric flask (6.7). Add 25ml of hydrochloric acid (5.4). Shake until a homogeneous suspensionis obtained, thenaddafurther 25ml of hydrochloric acid(5.4).
9.2.2Compensate for the acid consumption of the test portion (see9.1) by adding hydrochloric acid of a suitable
concentration sothat the volumeof thecontents of theflask is changedbynot morethan 1ml.
EXAMPLE Suppose that 5,0 ml of 0,1 mol/l hydrochloric acid is used in 9.1.2 for compensation of a sample rich in chalk, then
the acidconsumptionof the test portion is 0,5 mmol (9.1.3). Inthat case,add0,5 mlof 1,0 mol/lhydrochloricacid in9.2.2.
CAUTION — If the hydrochloric acid concentration in the suspension deviates from 0,31mol/l, a wrong
starch content will be determined. Too high or too low a hydrochloric acid concentration will result in toolow ortoo high values,respectively,of thedetermined starch content.
9.2.3Immerse the flask in the boiling water bath (6.3). During the first 3min, regularly shake the flask vigorously
toprevent clottingandtoobtainequal heat distributionin thesuspension.Whenshaking, keeptheflask immersed.
In the case of multiple analyses, immerse the flasks over suitable time intervals so as to keep the water bath at
boilingpoint.
After 15min /Gb15s, remove the conical flask from the bath. Immediately add 30ml of water (5.1) at a temperature
not exceeding10 /Gb0C andswirl. Cooltoa temperatureof about 20°C ina water bath withrunningcold water.
CAUTION — If the flask stays in the boiling water bath too long or if the temperature decrease is too slow,
toolow astarchcontent willbedetermined.
Add 5ml of potassium hexacyanoiron(II) solution (5.6.1) and shake for 1min. Add 5ml of zinc acetate solution
(5.6.2) and shake again for 1min. Dilute to the mark with water, mix and filter. Discard the first few millilitres of the
filtrate.
Determinethe optical rotationof thefiltrate ( /G61
1) withthe polarimeter or saccharimeter (6.4).
9.3Determination of opticalrotationofethanol-solublesubstances
9.3.1Weigh approximately 5g of the prepared test sample ( m2) to the nearest 1mg and transfer it quantitatively
to a dry 100ml volumetric flask (6.7). Add 40ml of ethanol (5.2). Shake until a homogeneous suspension isobtainedthen add afurther 40mlof ethanol(5.2).ISO 6493:2000(E)
© ISO2000 –All rights reserved 59.3.2Compensate for the acid consumption of the test portion (see9.1) by adding hydrochloric acid of a suitable
concentration so that the volume of the contents of the flask is changed by not more than 1ml. In principle, theamount of hydrochloric acidtobeaddedis twiceas muchas that added in 9.2.2.
9.3.3Shake vigorously and leave to stand for 1h at room temperature. During this time, shake vigorously at least
every10min.
If the lactose content of the sample exceeds 50g/kg (as for wheypowder and separated milk powder), dissolve the
samplebyheating the flask,fitted with areflux condenser, in a water bath at (50 /Gb12)°C for 30min.
Dilute to the mark with ethanol(5.2), mix and filter. Discard the first fewmillilitres of the filtrate.
Pipette 50ml of the filtrate into a 100ml volumetric flask (6.7). Add 2,0ml of hydrochloric acid (5.5) and swirl
vigorously. Fit areflux condenser totheflask andimmersetheflask inthe boiling water bath(6.3).
After 15min /Gb15s, remove the conical flask from the bath. Immediately add 30ml of water (5.1) at a temperature
not exceeding10 /Gb0C andswirl. Cooltoa temperatureof about 20°C in a water bathwithrunningcold water.
Add 5ml of potassium hexacyanoiron(II) solution (5.6.1) and shake for 1min. Add 5ml of zinc acetate solution
(5.6.2) and shake again for 1min. Dilute to the mark with water, homogenize and filter. Discard the first fewmillilitres of the filtrate.
Determinethe optical rotationof theclear filtrate( /G61
2) withthepolarimeter or saccharimeter (6.4).
10 Calculationandexpressionofresults
Calculate the starchcontent of thetest sample bythe equation:
wmm/G3d/Gb4 /G2dL
NMO
QP20000 25 5
201
12
2 /G61/G61/G61
D,
where
wis the numericalvalue of thestarchcontent of thetest sample, ingrams perkilogram;
/G611is the numericalvalue of thetotal optical rotation, indegrees, measuredin9.2;
/G612is the numerical value of the optical rotation, indegrees, of the ethanol-soluble substances measured in
9.3;
m1is the numerical value of the mass, ingrams, of the test portion for the determination of the total optical
rotation(9.2);
m2is the numerical value of the mass, ingrams, of the test portion for the determination of the optical rotation
of theethanol-solublesubstances (9.3);
/G61D20is the numerical value of the specific optical rotation, indegrees, of pure starch measured at a wavelength
of 589,3nm (NaD line):
/G61D20=185,9 for ricestarch;
/G61D20=185,7 for potatostarch(see annexA);
/G61D20=184,6 formaizestarch;ISO 6493:2000(E)
6 © ISO2000 –All rights reserved/G61D20=184,0 forryestarch;
/G61D20=183,6 for tapiocastarch(seeannexA);
/G61D20=182,7 for wheatstarch;
/G61D20=181,5 for barleystarch;
/G61D20=181,3 for oatstarch;
/G61D20=184,0 for other types of starchand starchmixtures inanimalfeedingstuffs.
Roundtheresultto the nearest1g/kg.
11 Precision
11.1 Interlaboratorytest
Details of an interlaboratory test on the precision of the method are given in annex B. The values derived from this
test maynot beapplicable toconcentration ranges andmatrices other thanthose given.
11.2 Repeatability
The absolute difference between two independent single test results, obtained using the same method on identicaltest material in the same laboratory by the same operator using the same equipment within a short interval of time,
willinnot morethan 5% of cases exceedtherepeatabilitylimit rgiven inor derivedfrom Table1.
Table1—Repeatabilitylimit (r)and reproducibilitylimit (R)
Sample Starch content
g/kgr
g/kgR
g/kg
Maize glutenmeal 190,4 12,5 20,2
Piglet feed 347,1 12,7 27,5
Layer feed 367,1 9,7 13,3
Peas 444,3 52,1 67,1
Dehydratedtapioca 629,3 15,0 36,1
11.3 Reproducibility
The absolute difference between two single test results, obtained using the same method on identical test material
in different laboratories by different operators using different equipment, will in not more than 5% of cases exceed
the reproducibilitylimit Rgiven inor derivedfrom Table1.ISO 6493:2000(E)
© ISO2000 –All rights reserved 712 Testreport
Thetest report shallspecify:
/Gbeall information necessaryfor the complete identification of the sample;
/Gbethesamplingmethodused, if known;
/Gbethetest methodused, withreferencetothis International Standard;
/Gbeall operating details not specified in this International Standard, or regarded as optional, together with details of
anyincidents whichmayhave influenced the test result(s);
/Gbethetest result obtained, or thetwo test results obtained if therepeatabilityhas beenchecked.ISO 6493:2000(E)
8 © ISO2000 –All rights reservedAnnex A
(informative)
Explanation ofthe specific opticalrotation ofpotato starch andtapioca
starch
A.1 Potato starch
For potatostarch, three different values of thespecific optical rotation appear tobeusedinpractice.
In the original publication by Ewers (see reference [2] in the Bibliography) two values are presented for the specific
opticalrotation of potatostarch:
/G61D20=185,7°for treatment of thestarchsampleina boiling water bath with 0,31mol/l hydrochloric acid;
/G61D20=195,4°for treatment of thestarchsampleina boiling water bath with0,10mol/l hydrochloric acid.
The polarimetric determination of starch content according to reference [2] with 0,10mol/l hydrochloric acid has
not become standard practice. Usually, for the determination of starch content 0,31mol/l hydrochloric acid is used
with the corresponding specific opticalrotation of 185,7°.
Unfortunately, in the past these two values from the original publication [2] have been confused in publications of
the European Community(EC) (see Bibliography) mentioning the specific optical rotation of potato starch. This hascausedconfusion.
In the Official Journal of the European Communities in 1967 (reference [3]), the correct value of 185,7° was
presented. After revision of the ECprotocol in 1972, the wrong value of 195,4° was mentioned in the Official
Journal [4].
In 1980, in the Official Journal [5], an incorrect rectification of the specific optical rotation of potato starch was
published: 195,4° was changed to185,4°. However, the latter should have been185,7°.
In 1987, the incorrect value of 195,4° introduced by the EC was adopted by the Analytical Working Party of the
Starch Experts Group(STEX) of theEuropean StarchAssociation(ESA) inreference[6].
Afterwards, the value wrongly rectified by the EC as 185,4°, was adopted in ISO/CD10520. In the subsequent
ISO/DIS10520 (1994) the correct value of 185,7° was presented. Although the wrong value of 185,4° wasintroduced again in the second ISO/DIS10520.2 (1995), the published version of ISO10520:1997 [7] contains thecorrect value of 185,7°.
A.2 Tapioca starch
Thevalueof 183,6°for thespecific opticalrotation of tapiocastarch has been adopted from reference[8].ISO 6493:2000(E)
© ISO2000 –All rights reserved 9Annex B
(informative)
Results ofinterlaboratorytest
The precision of the method was established in 1997 by an interlaboratory test carried out in accordance with
ISO 5725-2[10]. In this test 15laboratories participated. Samples were investigated of dehydrated tapioca, layer
feed, maize gluten meal, peas and piglet feed.
TableB.1gives asummaryof thestatisticalresults of the test.
TableB.1— Statistical results
Samplea
Parameter
12345
Number of laboratories retained after eliminating
outliers15 15 14 15 15
Number of acceptedresults 30 30 28 30 30
Mean starchcontent,g/kg 190,4 347,1 367,1 444,3 629,3
Repeatabilitystandard deviation( sr),g/kg 4,5 4,6 3,5 18,6 5,4
Repeatabilitycoefficient of variation,% 2,4 1,3 0,9 4,2 0,9
Repeatabilitylimit ( r)[r=2 , 8 /Gb4sr],g/kg 12,6 12,7 9,7 52,1 15,0
Reproducibilitystandard deviation ( sR),g/kg 7,2 9,8 4,2 24,0 12,9
Reproducibilitycoefficient of variation,% 3,8 2,8 1,3 5,4 2,0
Reproducibilitylimit ( R)[R=2,8 /Gb4sR],g/kg 20,2 27,5 13,3 67,1 36,1
a1: maize gluten meal;
2: piglet feed;
3: layerfeed;
4: peas;
5: dehydrated tapioca.ISO 6493:2000(E)
10 © ISO2000 –All rights reservedBibliography
[1] ISO 6497, Animalfeeding stuffs — Sampling .
[2] Ewers , Zeitschrift für öffentlicheChemie ,14,1908, pp. 150-157.
[3]OfficialJournal of the European Communities 2927/67of 1967-06-30.
[4]OfficialJournal of the European Communities L123/6-9of 1972-05-29.
[5]OfficialJournal of the European Communities L320/43of 1980-11-27.
[6] Stärke, 12(7), 1984, pp. 14-416.
[7] ISO 10520:1997, Native starch— Determinationof starchcontent — Ewers polarimetric method .
[8] DIN 10300:1971, Bestimmung des Rohstärkegehaltes— Salzsäure-Verfahren (Determination of crude
starchcontent- Hydrochloric acidmethod) .
[9] ISO 5725-1:1994, Accuracy (trueness and precision) of measurement methods and results — Part1:
General principles and definitions .
[10] ISO 5725-2:1994, Accuracy (trueness and precision) of measurement methods and results — Part2: Basic
methodfor the determination of repeatability andreproducibility of a standardmeasurement method .ISO 6493:2000(E)
ICS 65.120
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© ISO2000 –All rights reserved